Describe about PCR Reaction

1. Polymerase Chain
Reaction

2. Introduction
• PCR, polymerase chain reaction, is an in-
vitro technique for amplification of a region
of DNA whose sequence is known or which
lies between two regions of known
sequence
• Before PCR, DNA of interest could only be
amplified by over-expression in cells and
this with limited yield

3. • 1966, Thomas Brock discovers Thermus
aquaticus, a thermostable bacteria in the hot
springs of Yellowstone National Park
• 1983, Kary Mullis postulated the concept of
PCR ( Nobel Prize in 1993)
• 1985, Saiki publishes the first application of
PCR ( beta-Globin)
• 1985, Cetus Corp. Scientists isolate
Thermostable Taq Polymerase (from
T.aquaticus), which revolutionized PCR

4. Reaction Components
• DNA template
• Primers
• Enzyme
• dNTPs
• Mg2+
• buffers

5. 1- DNA template
• DNA containing
region to be
sequenced
• Size of target DNA
to be amplified : up
to 3 Kb

6. 2- Primers
• 2 sets of primers
• Generally 20-30
nucleotides long
• Synthetically
produced
• complimentary to the
3’ ends of target DNA
• not complimentary to
each other

7. Primers (ctnd)
• Not containing inverted repeat sequences to
avoid formation of internal structures
• 40-60% GC content preferred for better
annealing
• Tm of primers can be calculated to
determine annealing T0
• Tm= .41(%G+C) + 16.6log(J+) + 81.5
where J+ is the concentration of monovalent
ions

8. 3-Enzyme
• Usually Taq Polymerase or anyone of the
natural or Recombinant thermostable
polymerases
• Stable at T0 up to 950 C
• High processivity
• Taq Pol has 5’-3’ exo only, no proofreading

9. The PCR Cycle
• Comprised of 3 steps:
- Denaturation of DNA at 950C
- Primer hybridization ( annealing) at 40-
500C
- DNA synthesis ( Primer extension) at 720C

15. Standard thermocycle